PROJECT SUMMARY/ABSTRACT The bacillus Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis (TB) in humans. Despite a steady decline in TB mortality rates, multidrug resistant TB (MDR-TB) and relapse cases have increased. These observations highlight the urgent yet unmet need to understand how MDR-TB develops and persists. Moreover, new strategies to diagnose and treat Mtb infection can limit the spread of the disease and therefore the spread of resistance. Despite these needs, the mechanisms by which Mtb evades treatment remain insufficiently defined, in part due to a lack of tools available to probe Mtb physiology. Strategies that could mitigate these problems include early diagnosis, an understanding of variation in division rate between cells, and insight into Mtb persistence. Distinguishing phenotypic heterogeneity is difficult using traditional transcriptomics; chemical biology can yield tools to detect mycobacteria and monitor the dynamics of cell division. We have developed a fluorogenic probe (QTF) that reports on mycobacterial cell envelope assembly in real time. I shall employ QTF to identify and characterize distinct spatial and temporal profiles of bacteria from the Corynebacterineae suborder. Because QTF is activated by mycolyltransferase activity, fluorescence kinetics will be used for diagnostic purposes to identify and separate mycobacteria and corynebacteria in mixed microbial communities. I will also evaluate QTF in an isogenic monoculture, as I postulate this probe can be used to separate normal from persister cells. Finally, I shall develop a new fluorogenic probe, complementary to QTF, that will covalently label the growing cell wall. I envision QTF and this probe can simultaneously monitor mycolyltransferase activity cell envelope construction and remodeling.